ANALYTICAL
BIOCHEMISTRY
65, 362-368
Derivative
Absorption
(1975)
Spectrophotometry
of Native
Proteins
AYAKO ~ATSUS~IMA~*, YORINAO INOUE* AND KAZUO SHIBATA* The Institute of Physical and Chemical Research, Wake-shi, Saitama* and Tokyo Institute of Technology, Ookayama, Meguro-ku. Tokyo**, Japan
Received October
16, 1974; accepted December
1I. 1974
Derivative absorption spectrophotometry was applied to amino acids and native proteins (insulin. ribonuciease A, iysozyme and ~-chymotrypsin) in order to examine this technique as a tool to analyze aromatic amino acid residues in native proteins. The derivative spectrum of phenyialanine showed many sharp positive and negative bands between 230 and 270 nm while that of tyrosine or tryptophan showed only two or three negative bands between 270 and 300 nm, respectively. The derivative bands of these amino acid residues in native proteins shifted by a few nanometers toward the red. The states of these amino acid residues in several proteins were discussed from these shifts. It was concluded that the derivative absorption spectrophotometry is a useful technique for studying various states of phenyialanine residues in native proteins.
Ultraviolet absorption spectrophotometry has been applied to differentiate various states of aromatic amino acid residues in native proteins from the band positions and shapes of absorption bands. Such analysis by common absorption spectrophotometry has limitation because of overlapping of various strong and weak bands. Mutual overlappings of bands shift the maximum wavelengths, and strong bands such as those of tryptophan and tyrosine mask the weak bands of phenylalanine. Tyrosine and tryptophan residues in native proteins have, therefore, been studied extensively by this common absorption spectrophotometry or by difference spectrophotometry, but phenylalanine residues with weak absorption bands have been studied to a much lesser extent. French (1) stressed the importance of derivative absorption spectrophotometry as a tool to detect a weak band masked by a strong absorption band, provided that the derivative of absorbance, dEldX, with respect to wavelength for the weak band is appreciable. Derivative spectrophotometry has since been applied to various substances, in particular, for analysis of photosynthetic pigments such as chlorophylls and carotenoids in various states in vivo (2,3). Recently, Inoue er al. (4) developed a new technique to measure derivative spectra accurately and easily with a cassette-tape recorder 362 Copyright @ 1975 by Academic Press, Inc. Al1 rights of reproduction in any form resetwd.
DERIVATIVE
SPECTRA
OF
PHE
RESIDUES
363
connected to a spectrophotometer. In the circumstances that absorption spectrophotometry has been applied mostly to tryptophan and tyrosine residues but little to phenylalanine residues in proteins, this simple technique was applied in the present study to aromatic amino acids and several native proteins to examine its applicability. MATERIALS
AND
METHODS
Tyrosine, tryptophan and phenylalanine were purchased from Tokyo Kasei Kogyo Co., and crystalline bovine zinc insulin, lysozyme (egg white, 5 X crystallized) and ribonuclease A (bovine pancreas, 5 X crystallized) from Shimizu Pharmaceutical Co., Seikagaku Kogyo Co. and Worshington Biochemical Corp., respectively. a-Chymotrypsin was obtained from bovine pancreas by the method of Kunitz and Northrop (5). The B chain of insulin was prepared by the method of Kotaki (6) and purified by gel filtration through a Sephadex G-l 5 column. The concentrations of amino acids and proteins were determined spectrophotometrically, assuming the following values of E (molar extinction coefficient); 5.6 X lo3 M-‘cm-’ at 279 nm for tryptophan (7), 1.42 X lo3 M-‘cm-’ at 274.6 nm for tyrosine (7). 1.97 x 10” M-‘crn-’ at 257.4 nm for phenylalanine (7), 6.1 X IO3 M-‘cm-’ at 278 nm for insulin (S), 3.1 X 10” M-‘cm-’ at 276 nm for B chain of insulin (9), 3.88 X IO” M-‘cm-’ at 281 nm for lysozyme (IO), 9.8 X 10” M-‘cm-’ at 277.5 nm for ribonuclease A (11) and 5.0 x lo4 M-‘cm-’ at 280 nm for a-chymotrypsin ( 12). Absorption spectra of protein and amino acid solutions were measured with a Shimadzu UV-200 spectrophotometer. The derivative absorption spectra were measured with the same spectrophotometer by a technique developed in our laboratory (4). A cassette data recorder (Yasec model 110) was modified to memorize the same absorption spectrum on two separate parallel channels of a magnetic tape as frequency modulated (FM) analog signals with a slight shift (1 nm) in wavelength, and then the difference between the output signals from these channels was recorded on a chart in a two- or fivefold expanded scale. The wavelength shift of 1 nm was small enough to approximate the difference in the output to be the derivative of absorbance with respect to wavelength. RESULTS AND
The absorption (solid curves) of water are shown trum of tyrosine negative peak at
DISCUSSION
spectra (broken curves) and their derivative spectra tyrosine, tryptophan and phenylalanine dissolved in in Fig. la, b and c, respectively. The derivative specshows a strong negative peak at 285 nm and a weak 278 nm. The height in &ldh of the 285-nm peak is
364
MATSUSHIMA,
INOUE
AND
SHIBATA
+0.2 li (01 1 I,‘\ +01 , ’1II 0A 2 0 I- ,-’ +I ‘i’ I ’ -0.1
EF u
li
OB
0.6 E a4
; \
240zeJ2603300
240260260300 27024omaJ Wwe!enJth(nm)
FIG. 1. Absorption spectra (dashed line) and their derivative spectra (solid line) of tyrosine, tryptophan and phenylalanine. (a) 0.543 mM tyrosine. (b) 0.160 mM tryptophan. (c) 3.83 mM phenylalanine. The sample amino acids were dissolved in 0.09 M Tris buffer.
-164 X lo7 iv-‘cm-“, obtained by Brand& and Kaplan (13) for acetyl-Ltyrosine ethyl ester. As is clear from Fig. lb, the derivative spectrum of tryptophan shows negative peaks at 273, 283 and 291 nm, and the value of deldh at 291 nm is -670 x lo7 M-‘cm-“. The derivative spectrum of phenylalanine shows minima at 243, 248, 253, 259, 265 and 269 nm, and maxima at 235, 240, 245, 251, 256, 263 and 267 nm, and the deldk value of the strong negative peak at 265 nm is -32 X lo7 M-‘cmP2. The E values and their derivatives of these amino acids in the 250-270 nm region for phenylalanine bands are listed in Table 1. As is well known, the E values of the tyrosine band in the 250-270 nm region are several times higher and the E values of the tryptophan band are 10 - 30 times TABLE MOLAR EXTINCTION AROMATIC AMINO
1
COEFFICIENTS (E) AND THEIR FIRST DERIVATIVES (de/&) ACIDS IN THE 250-270 NM REGION FOR PHENYLALANINE ABSORPTION
BANDS
Tyrosine
Tryptophan
A Inm)
EU
deidhb
E
251 253 256 259 263 265 267 269
267
i27 +32 +41 +50 +58 i57 +50 +42
2,340
401 534 891 1.140
n M-’ cm-‘. * in 1 X 10’ Mm1cmm2.
OF
3,087 3.605 6,427 5,120
Phenylalanine drldh
+ 135 + 153 +169 +1s2 +169 +144 +110 + 78
E
deldA
143 148 156 172 142 120 85 52
+21 -9 +28 -27 +15 -32 -7 -25
DERIVATIVE
SPECTRA
OF
PHE
RESIDUES
365
-0.10 240 260 280 300 320 340 Wavelengthbx-nl
FIG. 2. Derivative spectra chain of insulin (lower figure) (curves B and D), respectively.
obtained in 0.09
for M Tris
488 PM tyrosine buffer at pH
(upper 7 (curves
figure) and 291 PM B A and C) and pH 12
higher than those of the phenylalanine bands. This great discrepancy between phenylalanine and tyrosine or tryptophan becomes much smaller when we compare their dcldh values. This makes it easier to identify phenylalanine as its derivative bands. In addition, the de/dA value for phenylalanine varies greatly with wavelength from a positive to a negative value or vice versa in a narrow wavelength region, while the deldh value of tyrosine or tryptophan in the same spectral region does not change much with wavelength. This makes it even easier for the identification. These two advantages are clearly seen in the data on native proteins presented below. The derivative spectra of insulin and its B chain and ribonuclease A which are devoid of tryptophan residues are shown in Figs. 2 and 3. These figures include the derivative spectra of these proteins and tyrosine as the reference at pH 7 (curves A and C) and pH 12 (curves B and D). The derivative spectrum of the B chain of insulin shows the strong
-0.10 240 260 280 X0
320 340
Wavelength
FIG. 3. Derivative spectra ribonuclease A (lower figure) (curves B and D), respectively.
obtained for 148 PM insulin (upper in 0.09 M Tris buffer at pH 7 (curves
figure) and 75.0 PM A and C) and pH 12
366
MATSUSHIMA.
INOUE
AND
SHIBATA
negative band of tyrosine at 285 nm, the same wavelength as found for free tyrosine. The same band is located at a longer wavelength of 287 nm for native insulin and at 288 nm for ribonuclease A. The same extent of wavelength shift was found for the weaker negative peak for native insulin and ribonuclease A. Brandts and Kaplan (13) found these shifts of tyrosine derivative bands for insulin and ribonuclease by calculation and attributed the shifts to buried tyrosine residues. The derivative spectra of ionized tyrosine (curve B in Fig. 2) and the B chain of insulin at alkaline pH (curve D in Fig. 2) show a single negative peak at 306 nm. This contrasts with three negative peaks found at 276, 285 and 306 nm for insulin (curve B in Fig. 3) and at 281, 289 and 306 nm for ribonuclease A (curve D in Fig. 3). The central negative peak of these three is ascribed to the tyrosine residues which did not ionize at the alkaline pH. It seems, therefore, that the two tyrosine residues in the B chain of insulin are in a freely ionizable state, and that some of the tyrosine residues in native insulin and ribonuclease A are in a state not ionizable at the alkaline pH, probably, being bound or buried in an interior of the molecule, as reviewed previously ( 14-I 6). This deduction is compatible with previous observations that one of the total four tyrosine residues in the insulin molecule is instantaneously ionizable (17) and that two of them are less reactive to cyanuric fluoride (18). The data on ribonuclease A also agree with the observation that two and three of the total six tyrosine residues in the molecule are in the less reactive and less ionizable states (19,20), respectively. Curves A and C in Fig. 4 are the derivative spectra obtained for lysozyme and cY-chymotrypsin and curves B and D in the same figure are the derivative spectra of amino acid mixtures with the same composition as those in these proteins, respectively. Three distinct negative peaks
I
240 260 280 300 WovelenglhM
320 340
FIG. 4. Derivative spectra of lysozyme and a-chymotrypsin. Upper figure: derivative spectra of lysozyme (curve A) and amino acid mixture (curve B) with the same composition as that in lysozyme. Lower figure: derivative spectra of cu-chymotrypsin (curve C) and amino acid mixture (curve D) with the same composition as that in cY-chymotrypsin. The concentration of lysozyme and ru-chymotrypsin were 11.3 and 15.4 pM, respectively.
DERIVATIVE
SPECTRA TABLE
367
OF PHE RESIDUES 2
THE POSITIONS OF MAXIMA AND MINIMA IN THE DERIVATIVE SPECTRA PHENYLALANINE AND PHENYIALANINE RESIDUES IN PROTEINS
Sample B Chain of insulin Insulin Ribonuclease A Lysozyme cu-Chymotrypsin Phenylalanine in water Phenylalanine in 60% dimethylsulfoxide
OF FREE
Maxima (nm)
Minima (nm)
250,256,263.267 252.258.264,268 253,258,264.268 259.2653268 253,258,264,268 251.256.263.267 253.258,264.269
2533259.2653269 254.260.266.270 260.266.270 260.266.271 254,260.266,270 253,259.265.269 255.261.267.271
appeared at 277, 286 and 295 nm in the derivative spectrum of lysozyme and at 277, 286 and 295 nm in the spectrum of a-chymotrypsin. The negative derivative peak at the longest wavelength of these proteins, which derives from the tryptophan absorption band at 291 nm (broken curve in Fig. lb), is shifted by 4 nm toward the red as compared with the corresponding derivative band of free tryptophan in the derivative spectra (curves B and D) of amino acid mixtures. As described above, the derivative spectrum of phenylalanine has many sharp positive and negative bands between 230 and 270 nm. Similar bands were found for the four species of native proteins at slightly longer wavelengths (Figs. 2-4). For example, positive peaks were at 252,258,264 and 268 nm and negative peaks at 254,260,266 and 270 nm for insulin (curve A in Fig. 3). The positions of the negative and positive bands found for the five species of proteins including the B chain of insulin as well as phenylalanine dissolved in water and in 60% dimethylsulfoxide are summarized in Table 2. The peak positions for the B chain of insulin agree well with those obtained for phenylalanine dissolved in water. On the other hand, the derivative bands observed for insulin, ribonuclease A, lysozyme and a-chymotrypsin are located at l-2 nm longer wavelengths than the corresponding bands of phenylalanine in water but at the same positions as the bands of phenylalanine dissolved in 60% dimethylsulfoxide. Yanari and Bovey (21) observed similar shifts of the absorption bands of benzene toward longer wavelengths by increasing the refractive index of solvent. Some of the phenylalanine residues in the above native proteins may, therefore, be in an environment with a refractive index higher than that of water, probably, being buried in the macromolecules. It was demonstrated in the present study that the derivative absorption spectrophotometry is a powerful tool to detect the phenylalanine bands masked by the strong absorption bands of tyrosine and tryptophan. It is useful not only for such qualitative detection but also for
368
MATSUSHIMA,
INOUE
AND
SHIBATA
observation of the shifts of the phenylalanine bands. There exist a number of techniques applicable for differentiation of various states of tyrosine or tryptophan residues in proteins; spectrophotometric titration of tyrosine residues and chemical differentiation based on different reactivities toward reagents. Common absorption spectrophotometry and chemical modification techniques are, however, not useful to study phenylalanine residues having weak absorption bands and being less reactive than tyrosine and tryptophan residues. This stresses the usefulness of the derivative absorption spectrophotometry presented in this paper. ACKNOWLEDGMENTS The present study was supported by research grants on “Chlorophyll formation” and on “Photosynthetic Reactioncenters” given by the Ministry of Education and by a grant for the study of “Life Sciences” at the Institute of Physical and Chemical Research (Rikagaku Kenkyusho).
REFERENCES 1. French, C. S., and Church, A. B. (1955) Carnegie Inst. Wash. Year Book 54, 162-165. 2. French, C. S., and Huang, H. S. (1957) Carnegie Inst. Wash. Year Book 56, 266-268. 3. French, C. S., and Elliot, R. E. (1958) Carnegie Inst. Wash. Year Book 57, 278-286. 4. Inoue, Y., Ogawa, T., Kawai, T., and Shibata, K. (1973) Physiol. Plant. 29, 390-395. 5. Kunitz, M., and Northrop, M. (1935) J. Gen. Physiol. 18, 433-458. 6. Kotaki, A. (1961)3. Biochem. (Tokyo) 50, 256-263. 7. Gratzer, W. B. (1970) in Handbook of Biochemistry, Selected Data for Biochemistry (H. A. Sober, ed.) 2nd ed., pp. B-74, Chemical Rubber Co., Cleveland, OH. 8. Weil, L., Seibles, T. S., and Herskovits, T. T. (1965) Arch. Biochem. Biophys. 111, 308-320. 9. Nakaya, K., Horinishi, H., and Shibata, K. (1967) J. Biochem. (Tokyo) 61, 34.5-351. 10. Fromageot, C., and Schneck, G. (1950) Biochim. Biophys. Acta 6, 113-122. 1 I. Bigelow, C. C. (1961) J. Biol. Chern. 236, 1706-1710. 12. Dixon, G. H., and Neurath, H. (1957)1. Biol. Chem. 225, 1049-1059. 13. Brandts, J. F., and Kaplan, L. J. (1973) Biochemistry 12, 201 l-2024. 14. Scheraga, H. A. (1961) Protein Structure, Academic Press, New York and London. 15. Joly, M. (1965) Physico-chemical Approach to the Denaturation of Proteins, Academic Press, New York and London. 16. Shibata, K. (197 1) in New Techniques in Amino Acids, Peptide, and Protein Analysis. (A. Niederwieser and G. Pataki, eds.) pp. 341-385. Ann Arbor Science Publisners, Ann Arbor, MI. 17. Inada, Y. (1961) J. Biochem. (Tokyo) 49, 217-225. 18. Kurihara, K.. Horinishi, H., and Shibata, K. (1963) Biochim. Biophys. Acta 74, 678-687. 19. Tanford, C., Hauenstein. J. D., and Rands, D. G. (1955) J. Amer. Chem. Sot. 77, 6409-64 13. 20. Takenaka. O., Horinishi. H., and Shibata, K. (1967)5. Biochem. (Tokyo) 62, 501-503. 21. Yanari, S., and Bovey, J. A. (1960)5. Biol. Chem. 235, 2818-2826.
BIOCHEMISTRY
65, 362-368
Derivative
Absorption
(1975)
Spectrophotometry
of Native
Proteins
AYAKO ~ATSUS~IMA~*, YORINAO INOUE* AND KAZUO SHIBATA* The Institute of Physical and Chemical Research, Wake-shi, Saitama* and Tokyo Institute of Technology, Ookayama, Meguro-ku. Tokyo**, Japan
Received October
16, 1974; accepted December
1I. 1974
Derivative absorption spectrophotometry was applied to amino acids and native proteins (insulin. ribonuciease A, iysozyme and ~-chymotrypsin) in order to examine this technique as a tool to analyze aromatic amino acid residues in native proteins. The derivative spectrum of phenyialanine showed many sharp positive and negative bands between 230 and 270 nm while that of tyrosine or tryptophan showed only two or three negative bands between 270 and 300 nm, respectively. The derivative bands of these amino acid residues in native proteins shifted by a few nanometers toward the red. The states of these amino acid residues in several proteins were discussed from these shifts. It was concluded that the derivative absorption spectrophotometry is a useful technique for studying various states of phenyialanine residues in native proteins.
Ultraviolet absorption spectrophotometry has been applied to differentiate various states of aromatic amino acid residues in native proteins from the band positions and shapes of absorption bands. Such analysis by common absorption spectrophotometry has limitation because of overlapping of various strong and weak bands. Mutual overlappings of bands shift the maximum wavelengths, and strong bands such as those of tryptophan and tyrosine mask the weak bands of phenylalanine. Tyrosine and tryptophan residues in native proteins have, therefore, been studied extensively by this common absorption spectrophotometry or by difference spectrophotometry, but phenylalanine residues with weak absorption bands have been studied to a much lesser extent. French (1) stressed the importance of derivative absorption spectrophotometry as a tool to detect a weak band masked by a strong absorption band, provided that the derivative of absorbance, dEldX, with respect to wavelength for the weak band is appreciable. Derivative spectrophotometry has since been applied to various substances, in particular, for analysis of photosynthetic pigments such as chlorophylls and carotenoids in various states in vivo (2,3). Recently, Inoue er al. (4) developed a new technique to measure derivative spectra accurately and easily with a cassette-tape recorder 362 Copyright @ 1975 by Academic Press, Inc. Al1 rights of reproduction in any form resetwd.
DERIVATIVE
SPECTRA
OF
PHE
RESIDUES
363
connected to a spectrophotometer. In the circumstances that absorption spectrophotometry has been applied mostly to tryptophan and tyrosine residues but little to phenylalanine residues in proteins, this simple technique was applied in the present study to aromatic amino acids and several native proteins to examine its applicability. MATERIALS
AND
METHODS
Tyrosine, tryptophan and phenylalanine were purchased from Tokyo Kasei Kogyo Co., and crystalline bovine zinc insulin, lysozyme (egg white, 5 X crystallized) and ribonuclease A (bovine pancreas, 5 X crystallized) from Shimizu Pharmaceutical Co., Seikagaku Kogyo Co. and Worshington Biochemical Corp., respectively. a-Chymotrypsin was obtained from bovine pancreas by the method of Kunitz and Northrop (5). The B chain of insulin was prepared by the method of Kotaki (6) and purified by gel filtration through a Sephadex G-l 5 column. The concentrations of amino acids and proteins were determined spectrophotometrically, assuming the following values of E (molar extinction coefficient); 5.6 X lo3 M-‘cm-’ at 279 nm for tryptophan (7), 1.42 X lo3 M-‘cm-’ at 274.6 nm for tyrosine (7). 1.97 x 10” M-‘crn-’ at 257.4 nm for phenylalanine (7), 6.1 X IO3 M-‘cm-’ at 278 nm for insulin (S), 3.1 X 10” M-‘cm-’ at 276 nm for B chain of insulin (9), 3.88 X IO” M-‘cm-’ at 281 nm for lysozyme (IO), 9.8 X 10” M-‘cm-’ at 277.5 nm for ribonuclease A (11) and 5.0 x lo4 M-‘cm-’ at 280 nm for a-chymotrypsin ( 12). Absorption spectra of protein and amino acid solutions were measured with a Shimadzu UV-200 spectrophotometer. The derivative absorption spectra were measured with the same spectrophotometer by a technique developed in our laboratory (4). A cassette data recorder (Yasec model 110) was modified to memorize the same absorption spectrum on two separate parallel channels of a magnetic tape as frequency modulated (FM) analog signals with a slight shift (1 nm) in wavelength, and then the difference between the output signals from these channels was recorded on a chart in a two- or fivefold expanded scale. The wavelength shift of 1 nm was small enough to approximate the difference in the output to be the derivative of absorbance with respect to wavelength. RESULTS AND
The absorption (solid curves) of water are shown trum of tyrosine negative peak at
DISCUSSION
spectra (broken curves) and their derivative spectra tyrosine, tryptophan and phenylalanine dissolved in in Fig. la, b and c, respectively. The derivative specshows a strong negative peak at 285 nm and a weak 278 nm. The height in &ldh of the 285-nm peak is
364
MATSUSHIMA,
INOUE
AND
SHIBATA
+0.2 li (01 1 I,‘\ +01 , ’1II 0A 2 0 I- ,-’ +I ‘i’ I ’ -0.1
EF u
li
OB
0.6 E a4
; \
240zeJ2603300
240260260300 27024omaJ Wwe!enJth(nm)
FIG. 1. Absorption spectra (dashed line) and their derivative spectra (solid line) of tyrosine, tryptophan and phenylalanine. (a) 0.543 mM tyrosine. (b) 0.160 mM tryptophan. (c) 3.83 mM phenylalanine. The sample amino acids were dissolved in 0.09 M Tris buffer.
-164 X lo7 iv-‘cm-“, obtained by Brand& and Kaplan (13) for acetyl-Ltyrosine ethyl ester. As is clear from Fig. lb, the derivative spectrum of tryptophan shows negative peaks at 273, 283 and 291 nm, and the value of deldh at 291 nm is -670 x lo7 M-‘cm-“. The derivative spectrum of phenylalanine shows minima at 243, 248, 253, 259, 265 and 269 nm, and maxima at 235, 240, 245, 251, 256, 263 and 267 nm, and the deldk value of the strong negative peak at 265 nm is -32 X lo7 M-‘cmP2. The E values and their derivatives of these amino acids in the 250-270 nm region for phenylalanine bands are listed in Table 1. As is well known, the E values of the tyrosine band in the 250-270 nm region are several times higher and the E values of the tryptophan band are 10 - 30 times TABLE MOLAR EXTINCTION AROMATIC AMINO
1
COEFFICIENTS (E) AND THEIR FIRST DERIVATIVES (de/&) ACIDS IN THE 250-270 NM REGION FOR PHENYLALANINE ABSORPTION
BANDS
Tyrosine
Tryptophan
A Inm)
EU
deidhb
E
251 253 256 259 263 265 267 269
267
i27 +32 +41 +50 +58 i57 +50 +42
2,340
401 534 891 1.140
n M-’ cm-‘. * in 1 X 10’ Mm1cmm2.
OF
3,087 3.605 6,427 5,120
Phenylalanine drldh
+ 135 + 153 +169 +1s2 +169 +144 +110 + 78
E
deldA
143 148 156 172 142 120 85 52
+21 -9 +28 -27 +15 -32 -7 -25
DERIVATIVE
SPECTRA
OF
PHE
RESIDUES
365
-0.10 240 260 280 300 320 340 Wavelengthbx-nl
FIG. 2. Derivative spectra chain of insulin (lower figure) (curves B and D), respectively.
obtained in 0.09
for M Tris
488 PM tyrosine buffer at pH
(upper 7 (curves
figure) and 291 PM B A and C) and pH 12
higher than those of the phenylalanine bands. This great discrepancy between phenylalanine and tyrosine or tryptophan becomes much smaller when we compare their dcldh values. This makes it easier to identify phenylalanine as its derivative bands. In addition, the de/dA value for phenylalanine varies greatly with wavelength from a positive to a negative value or vice versa in a narrow wavelength region, while the deldh value of tyrosine or tryptophan in the same spectral region does not change much with wavelength. This makes it even easier for the identification. These two advantages are clearly seen in the data on native proteins presented below. The derivative spectra of insulin and its B chain and ribonuclease A which are devoid of tryptophan residues are shown in Figs. 2 and 3. These figures include the derivative spectra of these proteins and tyrosine as the reference at pH 7 (curves A and C) and pH 12 (curves B and D). The derivative spectrum of the B chain of insulin shows the strong
-0.10 240 260 280 X0
320 340
Wavelength
FIG. 3. Derivative spectra ribonuclease A (lower figure) (curves B and D), respectively.
obtained for 148 PM insulin (upper in 0.09 M Tris buffer at pH 7 (curves
figure) and 75.0 PM A and C) and pH 12
366
MATSUSHIMA.
INOUE
AND
SHIBATA
negative band of tyrosine at 285 nm, the same wavelength as found for free tyrosine. The same band is located at a longer wavelength of 287 nm for native insulin and at 288 nm for ribonuclease A. The same extent of wavelength shift was found for the weaker negative peak for native insulin and ribonuclease A. Brandts and Kaplan (13) found these shifts of tyrosine derivative bands for insulin and ribonuclease by calculation and attributed the shifts to buried tyrosine residues. The derivative spectra of ionized tyrosine (curve B in Fig. 2) and the B chain of insulin at alkaline pH (curve D in Fig. 2) show a single negative peak at 306 nm. This contrasts with three negative peaks found at 276, 285 and 306 nm for insulin (curve B in Fig. 3) and at 281, 289 and 306 nm for ribonuclease A (curve D in Fig. 3). The central negative peak of these three is ascribed to the tyrosine residues which did not ionize at the alkaline pH. It seems, therefore, that the two tyrosine residues in the B chain of insulin are in a freely ionizable state, and that some of the tyrosine residues in native insulin and ribonuclease A are in a state not ionizable at the alkaline pH, probably, being bound or buried in an interior of the molecule, as reviewed previously ( 14-I 6). This deduction is compatible with previous observations that one of the total four tyrosine residues in the insulin molecule is instantaneously ionizable (17) and that two of them are less reactive to cyanuric fluoride (18). The data on ribonuclease A also agree with the observation that two and three of the total six tyrosine residues in the molecule are in the less reactive and less ionizable states (19,20), respectively. Curves A and C in Fig. 4 are the derivative spectra obtained for lysozyme and cY-chymotrypsin and curves B and D in the same figure are the derivative spectra of amino acid mixtures with the same composition as those in these proteins, respectively. Three distinct negative peaks
I
240 260 280 300 WovelenglhM
320 340
FIG. 4. Derivative spectra of lysozyme and a-chymotrypsin. Upper figure: derivative spectra of lysozyme (curve A) and amino acid mixture (curve B) with the same composition as that in lysozyme. Lower figure: derivative spectra of cu-chymotrypsin (curve C) and amino acid mixture (curve D) with the same composition as that in cY-chymotrypsin. The concentration of lysozyme and ru-chymotrypsin were 11.3 and 15.4 pM, respectively.
DERIVATIVE
SPECTRA TABLE
367
OF PHE RESIDUES 2
THE POSITIONS OF MAXIMA AND MINIMA IN THE DERIVATIVE SPECTRA PHENYLALANINE AND PHENYIALANINE RESIDUES IN PROTEINS
Sample B Chain of insulin Insulin Ribonuclease A Lysozyme cu-Chymotrypsin Phenylalanine in water Phenylalanine in 60% dimethylsulfoxide
OF FREE
Maxima (nm)
Minima (nm)
250,256,263.267 252.258.264,268 253,258,264.268 259.2653268 253,258,264,268 251.256.263.267 253.258,264.269
2533259.2653269 254.260.266.270 260.266.270 260.266.271 254,260.266,270 253,259.265.269 255.261.267.271
appeared at 277, 286 and 295 nm in the derivative spectrum of lysozyme and at 277, 286 and 295 nm in the spectrum of a-chymotrypsin. The negative derivative peak at the longest wavelength of these proteins, which derives from the tryptophan absorption band at 291 nm (broken curve in Fig. lb), is shifted by 4 nm toward the red as compared with the corresponding derivative band of free tryptophan in the derivative spectra (curves B and D) of amino acid mixtures. As described above, the derivative spectrum of phenylalanine has many sharp positive and negative bands between 230 and 270 nm. Similar bands were found for the four species of native proteins at slightly longer wavelengths (Figs. 2-4). For example, positive peaks were at 252,258,264 and 268 nm and negative peaks at 254,260,266 and 270 nm for insulin (curve A in Fig. 3). The positions of the negative and positive bands found for the five species of proteins including the B chain of insulin as well as phenylalanine dissolved in water and in 60% dimethylsulfoxide are summarized in Table 2. The peak positions for the B chain of insulin agree well with those obtained for phenylalanine dissolved in water. On the other hand, the derivative bands observed for insulin, ribonuclease A, lysozyme and a-chymotrypsin are located at l-2 nm longer wavelengths than the corresponding bands of phenylalanine in water but at the same positions as the bands of phenylalanine dissolved in 60% dimethylsulfoxide. Yanari and Bovey (21) observed similar shifts of the absorption bands of benzene toward longer wavelengths by increasing the refractive index of solvent. Some of the phenylalanine residues in the above native proteins may, therefore, be in an environment with a refractive index higher than that of water, probably, being buried in the macromolecules. It was demonstrated in the present study that the derivative absorption spectrophotometry is a powerful tool to detect the phenylalanine bands masked by the strong absorption bands of tyrosine and tryptophan. It is useful not only for such qualitative detection but also for
368
MATSUSHIMA,
INOUE
AND
SHIBATA
observation of the shifts of the phenylalanine bands. There exist a number of techniques applicable for differentiation of various states of tyrosine or tryptophan residues in proteins; spectrophotometric titration of tyrosine residues and chemical differentiation based on different reactivities toward reagents. Common absorption spectrophotometry and chemical modification techniques are, however, not useful to study phenylalanine residues having weak absorption bands and being less reactive than tyrosine and tryptophan residues. This stresses the usefulness of the derivative absorption spectrophotometry presented in this paper. ACKNOWLEDGMENTS The present study was supported by research grants on “Chlorophyll formation” and on “Photosynthetic Reactioncenters” given by the Ministry of Education and by a grant for the study of “Life Sciences” at the Institute of Physical and Chemical Research (Rikagaku Kenkyusho).
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